Erlotinib

For proliferation assay, BEAS2B cells stably expressing control or Mer (1 × 10³ cells per well) were cultured in the 6-well plates in medium containing full supplements. The cell proliferation was monitored every 3 days by cell counting with hemocytometer. For growth inhibition assay, cells (confluence 60–70%) were pretreated with Mer-specific inhibitor UNC569 (Selleck) or vehicle control (DMSO) for 12 h followed by incubation with serially diluted concentrations of erlotinib (Selleck) for 72 h. For experiments in PC9 cells, cells were directed with erlotinib without pretreatment procedure. At the termination of the experiment, 20 μl of MTT assay solution was added into 100 μl of medium containing cells and incubated for 2 h. The absorbance of each well was determined using a microplate reader (Molecular devices, Sunnyvale, CA) at 492 nm with reference wavelength at 630 nm. The percentage of cell survival was defined as the relative absorbance of untreated versus treated cells. All assays were performed in triplicate and repeated three times.

PC9 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China), and HCC827 cells were obtained from the American Type Culture Collection (Beijing, China). Both cell lines were exposed to high-dose (1–5 µM) erlotinib (Selleck Chem, Houston, USA) for a short time (72 h), and the surviving cells were continuously exposed to low doses of erlotinib (0.1 µM). This erlotinib administration approach was used for 8 months to establish resistant cell lines, PC9/ER and HCC827/ER, which were maintained by culturing in medium containing 1 µM erlotinib. The two resistant cell lines did not have a secondary T790M mutation, and the identity of all cell lines was confirmed by short tandem repeat (STR) profiling. All cells were maintained in RPMI-1640 medium (HyClone, Utah, USA) supplemented with 10% FBS (HyClone, Utah, USA) and 1% penicillin/streptomycin (Gibco, USA) at 37 °C in a humidified 5% CO₂ atmosphere.

The cell line HCC827 was purchased from ATCC. An erlotinib-resistant HCC827 cell line (HCC827ER) was generated as previously described [21 (link)]. Further, HCC827ER clones were established using minimal dilution [21 (link)]. All cells were maintained in RPMI 1640 media supplemented with 1% Penicillin-Streptomycin (Gibco), 1% Hepes 1 M buffer solution (Gibco), 1% Sodium Pyruvate (Gibco), 10% Fetal Bovine Serum (Gibco), and 1% 250 μg/mL Amphotericin B solution (Sigma-Aldrich). Further, the resistant cell line and clones were maintained in 5 μM erlotinib (Selleckchem).
For all mRNA and flow cytometry experiments cells were plated in 6-well plates and incubated for 24 h (300,000 cells/well). After incubation the cells were treated with drug and incubated for 72 h prior to harvest.
The inhibitors erlotinib, crizotinib, and SCH772984 were all purchased from Selleckchem.

We generated stable erlotinib-resistant cell lines as previously reported (17 (link)). In brief, erlotinib-resistant pancreatic cancer cell line AsPC-1/Erlo was developed from the AsPC-1 cell line by exposing the cells to increasing concentrations of erlotinib (Selleck Chemicals, Houston, TX, USA) for 6 months. Cells were cultured in drug-free media for 4-5 days before experiments to prevent acute drug effects.